Quality Control

Quality Control

All nucleic acids are subject to quality control in order to identify possible low-performing samples before they are used in protocols.

If samples do not meet our requirements for input amounts and/or concentration, you will receive an iLab notification detailing the QC findings and requesting your review of the data. Your project will be put on hold until permission to proceed is received. Via the comment section in your iLab request, you may request samples be omitted, ask to submit replacement samples, or choose to proceed with samples that have lower than required amounts and/or poor integrity.

Please note that the Advanced Genomics Core can almost always make a NGS library out of low and/or poor quality input amounts. These libraries will yield standard TapeStation traces and a nM concentration as measured by Kapa qPCR. If we see extremely low yields, excess adapters, or unusual profiles, you will be notified via iLab to determine if the libraries should be pulled from sequencing or subjected to additional processing (additional charge may apply). It is important to note that libraries generated from low and/or poor quality samples can exhibit lower complexity and an increased duplication rate which will be reflected in the sequencing data.

Nucleic acids isolated from FFPE samples will be poor quality. The core has modified protocols designed to maximize the chance of success, but you should be prepared for samples to drop out during processing. The more input you can provide for FFPE samples, the better the chance of success.

AGC DNA Quality Control includes:

  • Quantification using Qubit fluorescence-based assays
  • Visualization of DNA size on Agilent TapeStation

DNA traces for an intact genomic DNA with a DNA Integrity Number of 9.1 (left panel) and a degraded sample with a DNA Integrity number <2 (right panel)

 

AGC RNA Quality Control includes:

  • Quantification via Qubit fluorescence-based assays
  • Visualization of RNA size on Agilent TapeStation

    RNA Traces for a sample with a RNA Integrity Number of 10 (left panel) and a partially degraded sample with a RNA Integrity number of 5.6 (right panel)

 

AGC Library Quality Control includes:

  •   Quantification via Qubit
  •   Visualization of DNA size on Agilent TapeStation

Traces for final Illumina library (left panel) and a library requiring additional bead clean-up to remove excess free adapter (right panel)

  • Kapa real-time PCR to determine final concentration for sequencing

 

The Advanced Genomics Core accepts samples for quality control assessment only. Submissions go through iLab by initiating a Nucleic-Acid Quality Control request. For either DNA or RNA QC an aliquot of 3-5 µl of sample is required. DO NOT submit stock samples for analysis! Samples submitted for analysis will not be returned to researchers and will be discarded after 2 weeks. All samples are first subjected to Qubit Assay to determine concentration and inform selection of appropriate quality assay. All quality assessments samples will be done on the TapeStation, unless otherwise requested. Fees are charged per sample; you may be charged for additional samples if you are not running a full column (TapeStation) or cartridge (Bioanalyzer) and we do not have enough other samples to fill in the run.

Frequently Asked Questions

 

What options are available to check nucleic acid quality and quantity?
Quality Check Service Options Functional Range
Bioanalyzer DNA 1000 chip 100 pg/uL to 50 ng/uL
Bioanalyzer DNA 7500 chip 500 pg/uL to 50 ng/uL
Bioanalyzer DNA 12,000 chip 500 pg/uL to 50 ng/uL
Bioanalyzer DNA High Sensitivity chip (up to 7500 bp) 5 pg/uL to 500 pg/uL
Bioanalyzer RNA Nano chip (up to 6000 nt) 5 ng/uL to 500 ng/uL
Bioanalyzer RNA Pico chip (up to 6000 nt) 50 pg/uL to 5000 pg/uL
Bioanalyzer Small RNA chip (6 to 150 nt) 50 pg/uL to 5000 pg/uL
TapeStation DNA 1000 Tape (35 bp to 1000 bp) 100 pg/uL to 50 ng/ul
TapeStation DNA High Sensitivity 1000 Tape (35 bp to 1000 bp) 10 pg/uL to 1000 pg/uL
TapeStation Genomic DNA Tape (200 bp to > 60,000 bp) 5 ng/uL to 300 ng/uL
TapeStation RNA Tape (100 nt to 6000 nt) 25 ng/uL to 500 ng/uL
TapeStation High Sensitivity RNA Tape (100 nt to 6000 nt) 1 ng/uL to 25 pg/uL
Qubit Broad Range dsDNA Assay 0.5 ng/uL to 1000 ng/uL
Qubit High Sensitivity dsDNA Assay 10 pg/uL to 50 ng/uL
Qubit ssDNA Assay 50 pg/uL to 200 ng/uL
Qubit RNA Assay 1 ng/uL to 1000 ng/uL
Qubit High Sensitivity RNA Assay 250 pg/uL to 100 ng/uL
NanoDrop Spectrophotometer 10 ng/uL to 2500 ng/uL

For more information about each service option, refer the manufacturer’s websites for the Bioanalzyer & TapeStation and the Qubit & NanoDrop.

Why is my concentration different than that determined at the AGC?

There is notable variability between concentrations quantified spectrophotometrically (e.g. Nanodrop) as compared to fluorometrically (e.g. Qubit). While spectroscopic methods provide adequate estimation at concentrations higher than 10 ng/µl, fluorometric quantifications are more precise at lower concentrations. For this reason, all samples are quantified by Qubit upon entering the AGC.

How do I tell if gDNA is present in my RNA?

On an agarose gel, DNA contamination will be visible as a smear of band of fragments considerably larger than the RNA (>10 kb).  On Bioanalyzer or Tapestation RNA-chips, DNA will be visible in the size range from 4 to 10 kb.

What is the 260/280 ratio used for?

Nucleic acids and proteins have absorbance maxima at 260 and 280 nm, respectively. The ratio of absorbances at these wavelengths has historically been used as a measure of RNA or DNA “purity”. A ratio of ~1.8 is generally accepted as “pure” for DNA whereas a ratio of ~2.0 is preferred for RNA. Many samples fail to reach these minimum cutoffs. 260/280 ratios will fall below the acceptable limits if the sample concentration is low (<10ng/μl) or if the sample is contaminated by protein or other reagents such as phenol. Although not considered “pure”, these samples often perform well in downstream applications.

What is the 260/230 ratio used for?

The A260/A230 ratio is useful in determining the relative amounts of contaminants in purified nucleic acid samples. The presence of organic compounds in a sample will lead to a low A260/A230. Expected values for A260:A230 values are commonly 2.0-2.2. Samples with A260/A230 ratios outside of this range are considered to be low quality. However, many samples (e.g. biological fluids, FFPE, etc) rarely fall in this range. Nevertheless, these samples often perform well in downstream applications.

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